PCNA procedure posted as per request with notes - long

From:Robert Schoonhoven

There have been too many requests for me to fax them all out so I pasted
it into this e-mail.  This has been in several of our publications so
here it is. A few notes of explanation precede the actual procedure.

The PBSt referred to is PBS with 1.0% Tween 20  it actually does a few
things and one of them is the reduction of background.

I no longer use Innovex's Hematoxylin or DAB Enhancer.  For the DAB
enhancement I make up our own from a formula posted on the HistoNet some
time ago and it is as follows: 2.5 % CoCl2 with 2.0% Ni(NH4)2 in DDH2O.
For the hematoxylin I'd just use what you would normally use with your
IHC procedures, I now use Richard Allan's hematoxylin for 20 seconds.

DDH2O - I have the "luxury" of  having deionized, irradiated distilled
water.  I'm sure that deionized would be fine.

I'm not going to get into this in a lot of detail, but for the neophyte
to cell proliferation be aware that not all of the positive staining
that you see will be 'S' Phase cells.  Most (usually) will be G1 or G2
but they don't stain as darkly as those in 'S' do.  It take some
experience to make the determination but it's no rocket science.


PCNA Immunohistochemistry Protocol

1.  Deparaffinize and hydrate to distilled water.

2.  Antigen retrieval, if desired (separate protocol)

3.  DDH2O rinse

4.  Two 2-minute rinses with PBSt

5.  Peroxide block (DAKO Envision kit), 5 minutes

6.  Two 2-minute rinses with PBSt

7.  anti-PCNA (PC10), 1:100 dilution, 10 minutes

8.  Two 2-minute rinses with PBSt

9.  Labeled polymer (DAKO Envision kit), 10 minutes

10.  Two 2-minute rinses with PBSt

11.  DAB chromogen (DAKO Envision kit), diluted 1 drop/ml per mfr. SOP,
8 minutes.

12.  Rinse thoroughly with DDH2O

13.  DAB Enhancer solution - incubate for 5 minutes

14.  Rinse thoroughly with DDH2O

15.  Aqua Hematoxylin, 35 seconds.  Rinse thoroughly in tap water

16.  Blue in tap water, 5 minutes

17.  Dehydrate, clear, and coverslip.

Robert Schoonhoven

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